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首页> 外文期刊>Biochemistry >Catalytic activities and structural properties of horseradish peroxidase distal His42 --> Glu or Gln mutant
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Catalytic activities and structural properties of horseradish peroxidase distal His42 --> Glu or Gln mutant

机译：辣根过氧化物酶His42-> Glu或Gln突变体的催化活性和结构性质

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    The distal histidine (His) is highly conserved in peroxidases and has been considered to play a major role as a general acid-base catalyst for peroxidase reaction cycle. Recently, however, the X-ray structure of chloroperoxidase from the marine fungus Caldariomyces fumago has revealed that a glutamic acid is located at the position where most of the peroxidase has a histidine residue, suggesting that the carboxyl group in the glutamic acid (Glu) can also assist cleavage of an O-O bond in peroxides [Sundaramoorthy, M., Terner, J., & Poulos, T. L. (1995) Structure 3, 1367-1377]. In order to investigate catalytic roles of the glutamic acid at the distal cavity, two horseradish peroxidase mutants were prepared, in which the distal His42 has been replaced by Glu (H42E) or Gln (H42Q). The formation rate of compound I in the H42E mutant was significantly greater than that for the H42Q mutant, indicating that the distal Glu can play a role as a general acid-base catalyst. However, the peroxidase activity of the H42E mutant was still lower, compared to that for native enzyme. On the basis of the CD, resonance Raman, and EPR spectra, it was suggested that the basicity of the distal Glu is lower than that of the distal His and the position of the distal Glu is not fixed at the optimal position as a catalytic amino acid residue, although no prominent structural changes around heme environment were detected. The less basicity and improper positioning of the distal Glu would destabilize the heme-H2O2-distal Glu ternary intermediate for the peroxidase reaction. Another characteristic feature in the mutants was the enhancement of the peroxygenase activity. Since the peroxygenase activity was remarkably enhanced in the H42E mutant, the distal Glu is also crucial to facilitate the peroxygenase activity as well as the enlarged distal cavity caused by the amino acid substitution. These observations indicate that the distal amino acid residue is essential for function of peroxidases and subtle conformational changes around the distal cavity would control the catalytic reactions in peroxidase.
    机译：远端组氨酸（His）在过氧化物酶中高度保守，并被认为是过氧化物酶反应周期中通用的酸碱催化剂起着主要作用。然而，最近，来自海洋真菌Cumdariamyces fumago的氯过氧化物酶的X射线结构表明，谷氨酸位于大多数过氧化物酶具有组氨酸残基的位置，这表明谷氨酸（Glu）中的羧基还可在过氧化物中切割OO键[Sundaramoorthy，M.，Terner，J。，＆Poulos，TL（1995）Structure 3，1367-1377]。为了研究谷氨酸在远端腔中的催化作用，制备了两个辣根过氧化物酶突变体，其中远端His42已被Glu（H42E）或Gln（H42Q）取代。 H42E突变体中化合物I的形成速率显着大于H42Q突变体中化合物I的形成速率，表明远端Glu可以充当一般的酸碱催化剂。但是，与天然酶相比，H42E突变体的过氧化物酶活性仍然较低。根据CD，共振拉曼光谱和EPR光谱，建议远端Glu的碱性低于远端His的碱性，并且远端Glu的位置未固定在作为催化氨基的最佳位置酸残基，尽管在血红素环境周围未发现明显的结构变化。远端Glu的碱度较低和定位不当会使过氧化物酶反应的血红素-H2O2-远端Glu三元中间体不稳定。突变体的另一个特征是过氧化酶活性的增强。由于过氧化酶活性在H42E突变体中显着增强，因此远端Glu对于促进过氧化酶活性以及由氨基酸取代引起的远端腔增大也至关重要。这些观察结果表明，远端氨基酸残基对于过氧化物酶的功能是必不可少的，并且远端腔周围的细微构象变化将控制过氧化物酶中的催化反应。

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